Cloning vectors are important to understanding and manipulating various cellular processes and the underlying biochemical pathways. Such understanding enriches scientific knowledge and helps lead to new discoveries. Ultimately, such discoveries can lead to the development of valuable research tools and effective therapeutic compositions and treatments.
In stark contrast to the many vectors capable of manipulating short sequences, very few cloning vectors have been developed as tools for the genetic analysis, engineering and delivery of large nucleic acid sequences (e.g., entire genomes). The cloning and manipulation of complex sequences is inherently difficult due to the length of the inserts, which adversely affects the efficiency of the ligation reactions. Also, the scarcity of unique restriction sites further limits preparing large nucleic acids.
Those systems optimized for the analysis and manipulation of large nucleic acid sequences to date are ineffective for the delivery of such sequences to a target cell. Systems optimized for the delivery of large sequences also have been inefficient for analysis/manipulation of nucleic acids. For example, to prepare viral vectors containing a foreign gene, Stratford-Perricaudet et al. (J. Clin. Invest. 90:626–630, 1992) teach the use of homologous recombination to generate recombinant viruses in mammalian packaging cell lines.
Of the commonly used viral vectors, lentiviruses can be difficult to propagate and are relatively small (˜9 kb). Thus, such vectors suffer from propagation difficulties and limited insert length capacity. (Wivel et al. (1998) Hematol. Oncol. Clin. North Am. 12(3):483–501). A limited insert size has the added drawback of perhaps preventing the addition of regulatory sequences.
Similarly, AAV-based vectors, because of relatively small size (˜4.5 kb) are limited greatly in the maximum insert size. (Flotte et al. (1995) Gene Ther. 2(6):357–362).
Adenovirus-based vectors offer several attractive features including ease of propagation, high level of transgene expression, lack of integration in the host genome, which lowers the risk of mutagenesis, and the ability of carrying larger inserts (˜35 kb) (Hardy et al. (1997) J. Virol. 71(3):1842–1849).
Some efforts to develop recombinant adenoviruses employed three different approaches that rely on homologous recombination in either mammalian cells, yeast cells or bacterial cells.
Homologous recombination in mammalian cells is the most widely applied. Mittal et al. (Virus Research 28:67–90, 1993) teach the co-transfection of two plasmids containing a split defective genome into a complementary packaging cell line capable of rescuing the defective adenovirus.
However, as noted above, homologous recombination in mammalian cells is a rare event. Thus, the use of mammalian cells can be inefficient. The mammalian cell approach also requires repeated rounds of plaque purification as well as complex and time consuming viral production protocols. In addition, because the introduction of specific mutations in the regions of the vectors other than the ends of the adenoviral sequences is extremely tedious, engineering and recovering multiple mutations in the recombinant vector is virtually impossible.
Ketner et al. (Proc. Natl. Acad. Sci. 91:6186–6190, 1994) teach a yeast-based system in which the full length adenovirus genome was cloned and maintained as an infectious yeast artificial chromosome. That system relied on the high homologous recombination rate in yeast to modify any sequence within the vector and to introduce multiple inserts as needed. However, the low efficiency of formation of the recombinant vector and the low yield of the recombinant vector from yeast cells severely limit the ability to rescue virions, thus making transfection very difficult.
Attempts to overcome the limitations of the yeast-based system lead Chartier et al. (Virol. 70:48054810, 1996), Crouzet et al. (Proc. Natl. Acad. Sci. 94:1414–1419, 1997) and He et al. (Proc. Natl. Acad. Sci. 95:2509–2514, 1998) to develop bacterial systems. Bacterial systems offer the advantage of higher recombination rates and thus are more efficient.
However, those systems require large, cumbersome screening processes to identify recombinant clones. Other considerable limitations are the inability to engineer multiple mutational inserts and the need for highly specific bacterial shuttle vectors for each specific bacterial system.
Thus, there remains a yet unfulfilled need for versatile recombinatorial vectors and methods capable of overcoming the shortcomings of existing approaches. Such vectors and methods should be capable of broad targeting range of both dividing and non-dividing cells, and high levels of transgene expression. Ideally, such systems would have high recombination rates while minimizing the risk of integration in the host genome. To address present needs, such systems should allow the manipulation of large sequences and engineering multiple mutational inserts, while minimizing the need for extensive screening protocols. Such vectors also should be propagated easily and allow the recovery of sufficient amounts for the delivery of such recombinant nucleic acids directly to mammalian cells in vitro or in vivo.